GRP94, the endoplasmic reticulum Hsp90 chaperone, has been identified as a potent tumor rejection antigen, capable of eliciting vigorous immune responses against primary and distant metastases of its tumor of origin. The immunogenic activity of GRP94 is thought to derive from an as yet uncharacterized peptide binding activity. In eliciting an immune response, GRP94-peptide complexes are internalized by antigen presenting cells (APC) and the GRP94-bound peptides re-presented on the APC class I molecules for avtivation of peptide-specific cytotoxic T lymphocytes. The immunogenic properties of GRP94 are well established, and tumor-derived GRP94 is now in multi-center Phase III clinical trials as an immunotherapy for renal cell carcinoma. In addition to its activity as a tumor antigen, there now exists strong evidence that GRP94 functions as a protein second messenger in the communication of pathological cell death to the immune system. This conclusion extends from recent work demonstrating that GRP94 is released from cells in response to lethal viral infection and can subsequently be processed by APCs to elicit virus-specific immune responses. In pursuing research into the biological basis of GRP94 function, two primary long term objectives are proposed: 1) Define the structural and regulatory basis for GRP94's function as a peptide binding protein; 2) Define the mechanism of GRP94 uptake into, and subcellular fate within, antigen presenting cells. The proposed studies will draw on a variety of biophysical techniques, for the analysis of protein structure; biochemical techniques, to study peptide-GRP94 interactions; and cell biological/immunological techniques, to define the cellular basis for GRP94-dependent immune responses. The broad scope of techniques used in these studies is intended to allow a detailed molecular description of the structural and functional basis for the immunogenic activity of GRP94. Insights obtained in achieving the primary objectives of this proposal are expected to contribute significantly to the development of molecular chaperone proteins as immunotherapeutic agents for the treatment of cancer and infectious disease. In addition, results obtained in studies defining a role for GRP94 as a second messenger for pathological cell death will benefit the fields of human and animal vaccin4 development and the study of the genesis and therapeutic resolution of autoimmunity.